Classical swine fever (“CSF”) is a highly contagious disease of pigs and wild boar. Although it has been eradicated from many countries, CSF continues to cause serious problems in different parts of the world (Moenning et al., Clinical Signs And Epidemiology Of Classical Swine Fever: A Review Of New Knowledge. Vet. J. 2003, 165, 11-20; Edwards et al., Classical Swine Fever: The Global Situation, Vet. Microbiol. 2000, 73, 103-19). The causative agent, CSF virus (“CSFV”) is a member of the genus Pestivirus within the family Flaviviridae. The other members within the genus Pestivirus are bovine viral diarrhoea virus (“BVDV”) and border disease virus (“BDV”). The natural hosts for BVDV and BDV are cattle and sheep, respectively, but both viruses can naturally infect pigs also. Antibodies against BVD virus and BD virus may cross-react with CSFV in serological assays, and cause diagnostic problems (Terpstra, C., Wensvoort, G., A Congenital Persistent Infection Of Bovine Virus Diarrhoea Virus In Pigs: Clinical, Virological And Immunological Observations. Vet. Q., 1997, 19, 97-101; Paton, D. J., Done, S. H., Congenital Infection Of Pigs With Ruminant-Type Pestiviruses, J. Comp. Pathol. 1994, 111,151-63; Paton et al. Infection Of Pigs And Cattle With Bovine Viral Diarrhoea Virus On A Farm In England, Vet. Rec., 1992, 131, 185-8.). Because only CSFV is classified within the list A diseases of the Office International des Epizooties (“OIE”), it is important to differentiate between CSFV and BVDV or BDV.
Pestiviruses are small enveloped viruses containing a positive sense single stranded RNA of approximately 12.5 kb. Their genomes have a large open reading frame flanked by highly conserved 5′- and 3′-non-translated regions (“NTR”). In the last decade, mainly the 5′NTR served as the template for species and genus overlapping genome amplifications by using the RT-PCR (Vilcek et al., Pestiviruses Isolated From Pigs, Cattle And Sheep Can Be Allocated Into At Least Three Genogroups Using Polymerase Chain Reaction And Restriction Endonuclease Analysis. Arch. Virol. 1994 136, 309-23; Sandvik et al., Detection And Identification Of Ruminant And Porcine Pestiviruses By Nested Amplification Of 5′ Untranslated Cdna Regions. J. Virol. Methods, 1997, 64, 43-56.; Hyndman et al., A Novel Nested Reverse Transcription PCR Detects Bovine Viral Diarrhoea Virus In Fluids From Aborted Bovine Fetuses, J. Virol. Methods, 1998, 71, 69-76.; Paton et al., Classical Swine Fever Virus: A Ring Test To Evaluate RT-PCR Detection Methods, Vet. Microbiol,. 2000, 73, 159-74; Patton et al., Classical Swine Fever Virus: A Second Ring Test To Evaluate RT-PCR Detection Methods, Vet. Microbiol., 2000, 77, 71-81; Barlic-Maganja and Grom, Highly Sensitive One-Tube RT-PCR And Microplate Hybridisation Assay For The Detection And For The Discrimination Of Classical Swine Fever Virus From Other Pestiviruses, J. Virol. Methods, 2001, 95, 101-10). The classical RT-PCR proved to be a sensitive and specific diagnostic tool. However, detection of the amplified PCR products by gel-based systems bears the risk for cross-contaminations, does not allow exact quantification of genome copies in the template, and does not include an additional specificity test. The introduction of fluorogenic probes allows the detection of sequence specific templates achieved in real-time without opening the PCR tubes (Gibson et al., A Novel Method For Real Time Quantitative RT-PCR. Genome Res. 1996, 6, 995-1001 Heid et al., 1996). Since real-time PCR does not require post-PCR sample handling contaminations can be avoided, and the hybridisation of the probe ensures specificity.
For the diagnosis of Pestiviruses, TaqMan-probes proved to be practicable and robust and were used by several authors (McGoldrick et al., A Novel Approach To The Detection Of Classical Swine Fever Virus By RT-PCR With A Fluorogenic Probe (TaqMan). J. Virol. Methods, 1998, 72, 125-35; 1999; Bhudevi and Weinstock, Detection of bovine viral diarrhea virus in formalin fixed paraffin embedded tissue sections by real-time RT-PCR (Taqman). J. Virol. Methods, 2003, 109, 25-30; Bhudevi and Weinstock, Fluorogenic RT-PCR Assay (Taqman) For Detection And Classification Of Bovine Viral Diarrhea Virus. Vet. Microbiol. 2001, 83, 1-10.; Risatti et al., Rapid Detection Of Classical Swine Fever Virus By A Portable Real-Time Reverse Transcriptase PCR Assay, J. Clin. Microbiol. 2003, 41, 500-5). All currently described real-time RT-PCR assays for the detection of pestiviral sequences amplified targets within the 5′NTR gave results with acceptable sensitivity and specificity. However, no internal controls verifying the RNA isolation step as well as the RT-PCR have been used so far. In 2003, a multiplex real-time RT-PCR for the detection of CSFV was described in which a chimeric CSF virus containing the 5′NTR of BVDV as an universal positive control was used (Hofmann, Construction Of An Infectious Chimeric Classical Swine Fever Virus Containing The 5′UTR Of Bovine Viral Diarrhea Virus, And Its Application As A Universal Internal Positive Control In Real-Time RT-PCR. J. Virol. Methods, 2003, 114, 77-90). However, such a complete chimeric virion might be infectious and would therefore not be suitable for routine diagnostic purposes.
The present invention provides a robust ready to use, highly sensitive, and CSFV-specific multiplex real-time RT-PCR assay with two controls for the simple and fast routine diagnosis of CSF. The first control (positive control) proves the efficiency of primers and probes, while the second control (internal control) is designed to check RNA isolation and RT-PCR of each sample tested.